It is well-known to modify the properties and characteristics of proteins by conjugating groups to the protein in order to change the properties of the protein. In fact, more than twenty years ago, U.S. Pat. No. 4,179,337 taught proteins conjugated to polyethylene or polypropylene glycols. Generally, such conjugation generally requires some functional group in the protein to react with another functional group in a conjugating group. Typically, amino groups, such as the N-terminal amino group or the 8-amino group in lysines, have been used in combination with a suitable acylating reagent. It is often desired or even required to be able to control the conjugation reaction, i.e. to control where the conjugating compounds are attached and to control how many conjugating groups are attached. This is often referred to as specificity or selectivity.
However, the repertoire of selective chemical reactions is very limited. An alternative is, by recombinant methods, to introduce special unnatural amino acids having a unique reactivity and then exploit this reactivity in the further derivatization. An alternative is to use enzymes which recognize structural and functional features of the protein to be modified. An example of this is the use of microbial transglutaminase (mTGase) to selectively modify Gln residues in growth hormone. Specifically, transglutaminase has been used in the food industry and particular in the diary industry to cross-bind proteins. Other documents disclose the use of transglutaminase to alter the properties of physiologically active proteins. See e.g. EP 950665, EP 785276 and Sato, Adv. Drug Delivery Rev., 54, 487-504 (2002), which disclose the direct reaction between proteins comprising at least one Gln and amine-functionalised PEG or similar ligands in the presence of transglutaminase; see also Wada in Biotech. Lett., 23, 1367-1372 (2001), which discloses the direct conjugation of β-lactoglobulin with fatty acids by means of transglutaminase. The reaction catalysed by the transglutaminase is a transamidation reaction in which the primary amide of the glutamine residue is converted to a secondary amide from a primary amine present in the reaction mixture, as shown in FIG. 1.
However, selective derivatization of proteins is a very difficult task; the derivatization of lysines in a protein by acylation is an even more inherently non-selective process. Thus, there is at present no efficient method for the selective derivatisation of lysine residues. Accordingly, there is an immediate need in the art for methods of selectively derivatizing amino acid residues such as lysine in proteins or polypeptides of interest.